Cancer immunotherapy by radiofrequency electrical membrane breakdown (RF-EMB)

ABSTRACT

A method of non-thermally ablating undesirable tissue in the body by application of pulsed, bipolar, instant charge reversal electrical fields of sufficient energy to cause complete and immediate cell membrane rupture and destruction. Energy is delivered through radio frequency pulses of particular frequencies, wave characteristics, pulse widths and pulse numbers, such that enhanced physical stresses are placed on the cell membrane to cause its immediate and complete destruction thereby spilling the entire cell content and membrane constituents into, the extracellular space without denaturing proteins so as to enable an immunological response to destroy and remove the target tissue and similarly marked tissue elsewhere in the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication No. 61/912,172 filed Dec. 5, 2013 and titled “Cancer AntigenEnhanced Preservation to Antigen Presenting Cells by RadiofrequencyElectrical Membrane Breakdown (RF-EMB) as an Adjuvant Mechanism forImmunotherapy,” which is here incorporated in its entirety by reference.This patent application also claims priority to U.S. patent applicationSer. No. 14/451,333, filed Aug. 4, 2014, now U.S. Pat. No. 10,154,869,and tided “System And Method For Creating Radio-Frequency EnergyElectrical Membrane Breakdown For Tissue Ablation” which is hereincorporated in its entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of medical ablationof biological tissue for treatment of disease and, more particularly, tothe controlled application of radio frequency energy to soft tissue andcancerous tissue in humans and mammals to ablate such tissue throughcellular destruction by Electrical Membrane Breakdown.

2. Description of the Background

Cancer is not one single disease but rather a group of diseases withcommon characteristics that often result in sustained cellproliferation, reduced or delayed cell mortality, cooption of bodilyangiogenesis and metabolic processes and evasion of bodily immuneresponse which results in undesirable soft tissue growths calledneoplasms or, more commonly, tumors. Removal or destruction of thisaberrant tissue is a goal of many cancer treatment methods andmodalities. Surgical tumor excision is one method of accomplishing thisgoal. Tissue ablation is another, minimally invasive method ofdestroying undesirable tissue in the body, and has been generallydivided into thermal and non-thermal ablation technologies. Thermalablation encompasses both the addition and removal of heat to destroyundesirable cells. Cryoablation is a well established technique thatkills cells by freezing of the extracellular compartment resulting incell dehydration beginning at −15 C and by intracellular ice formationcausing membrane rupture occurring at colder temperatures. Becausecryoablative techniques can rupture the cell membrane without denaturingcell proteins under certain conditions, such techniques have theadditional ability to stimulate an antitumor immune response in thepatient.

Heat based techniques are also well established for ablation of bothcancerous and non cancerous tissues and include radio-frequency (RF)thermal, microwave and high intensity focused ultrasound ablation whichraise localized tissue temperatures well above the body's normal 37° C.These methods use various techniques to apply energy to the target cellsto raise interstitial temperature. For example, RF thermal ablation usesa high frequency electric field to induce vibrations in the cellmembrane that are converted to heat by friction. Cell death occurs in aslittle as 30 second once the cell temperature reaches 50° C. anddecreases as the temperature rises. At 60° C. cell death isinstantaneous. If the intracellular temperature rises to between about60 and 95° C., the mechanisms involved in cell death include cellulardesiccation and protein coagulation. When the intracellular temperaturereaches 100° C., cellular vaporization occurs as intracellular waterboils to steam. In the context of tissue ablation, cell temperatures notexceeding 50° C. are not considered clinically significant. Becausecellular proteins are denatured by the heat of thermal ablationtechniques, they are not available to stimulate a specific immuneresponse as they may be with cryoablation. Both heat based andcryoablation techniques suffer from the drawback that they have littleor no ability to spare normal structures in the treatment zone and socan be contraindicated based on tumor location or lead to complicationsfrom collateral injury.

Non thermal ablation techniques include electrochemotherapy andirreversible electroporation which although quite distinct from oneanother, each rely on the phenomenon of electroporation. With referenceto FIG. 1, electroporation refers to the fact that the plasma membraneof a cell exposed to high voltage pulsed electric fields within certainparameters, becomes temporarily permeable due to destabilization of thelipid bilayer and the formation of pores P. The cell plasma membraneconsists of a lipid bilayer with a thickness t of approximately 5 nm.With reference to FIG. 2A, the membrane acts as a nonconducting,dielectric barrier forming, in essence, a capacitor. Physiologicalconditions produce a natural electric potential difference due to chargeseparation across the membrane between the inside and outside of thecell even in the absence of an applied electric field. This restingtransmembrane potential V′m ranges from 40 mv for adipose cells to 85 mvfor skeletal muscle cells and 90 mv cardiac muscle cells and can vary bycell size and ion concentration among other things.

With continued reference to FIGS. 2B-2D, exposure of a cell to anexternally applied electric field E induces an additional voltage Vacross the membrane as long as the external field is present. Theinduced transmembrane voltage is proportional to the strength of theexternal electric field and the radius of the cell. Formation oftransmembrane pores P in the membrane occurs if the cumulative restingand applied transmembrane potential exceeds the threshold voltage whichmay typically be between 200 mV and 1 V. Poration of the membrane isreversible if the transmembrane potential does not exceed the criticalvalue such that the pore area is small in relation to the total membranesurface. In such reversible electroporation, the cell membrane recoversafter the applied field is removed and the cell remains viable. Above acritical transmembrane potential and with longer exposure times,poration becomes irreversible leading to eventual cell death due aninflux of extracellular ions resulting in loss of homeostasis andsubsequent apoptosis. Pathology after irreversible electroporation of acell does not show structural or cellular changes until 24 hours afterfield exposure except in certain very limited tissue types. However, inall cases the mechanism of cellular destruction and death by IRE isapoptotic which requires considerable time to pass and is not visiblepathologically in a time frame to be clinically useful in determiningthe efficacy of IRE treatment which is an important clinical drawback tothe method.

Developed in the early 1990's, electrochemotherapy combines the physicaleffect of reversible cell membrane poration with administration ofchemotherapy drugs such as cisplatin and bleomycin. By temporarilyincreasing the cell membrane permeability, uptake of non-permeant orpoorly permeant chemotherapeutic drugs is greatly enhanced. After theelectric field is discontinued, the pores close and the drug moleculesare retained inside the target cells without significant damage to theexposed cells. This approach to chemotherapy grew out of earlierresearch developing electroporation as a technique for transfection ofgenes and DNA molecules for therapeutic effect. In this context,irreversible electroporation leading to cell death was viewed as afailure in as much as the treated cells did not survive to realize themodification as intended.

Irreversible electroporation (IRE) as an ablation method grew out of therealization that the “failure” to achieve reversible electroporationcould be utilized to selectively kill undesired tissue. IRE effectivelykills a predictable treatment area without the drawbacks of thermalablation methods that destroy adjacent vascular and collagen structures.During a typical IRE treatment, one to three pairs of electrodes areplaced in or around the tumor. Electrical pulses carefully chosen toinduce an electrical field strength above the critical transmembranepotential are delivered in groups of 10, usually for nine cycles. Each10-pulse cycle takes about one second, and the electrodes pause brieflybefore starting the next cycle. As described in U.S. Pat. No. 8,048,067to Rubinsky, et. al and application Ser. No. 13/332,133 by Arena, et al.which are incorporated here by reference, the field strength and pulsecharacteristics are chosen to provide the necessary field strength forIRE but without inducing thermal effects as with RF thermal ablation.However, because cells ablated by IRE methods undergo apoptotic deathwithout membrane rupture their ability to induce a supplemental immuneresponse as observed with cryoablation is impaired. When used as thesole ablative tool in a treatment protocol, IRE's inability to induce asupplemental immune response is a substantial limitation to itstherapeutic benefit fir patients. On the other hand, cryoablationsuffers from the significant clinical disadvantages arising from theextreme cold and its capacity to destroy nearby critical healthystructures. What is needed is a minimally invasive tissue ablationtechnology that can avoid damaging healthy tissue while exposingcellular contents without denaturing such cellular contents so that theycan to trigger a clinically useful immune response.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide a methodof tissue ablation using electrical pulses which causes immediate celldeath through the mechanism of complete break down the membrane of thecell.

It is another object of the present invention to provide a method oftissue ablation that causes immediate cell death electrically breakingdown the cell membrane such that it can be monitored by immediatepathologic, chemical or spectroscopic examination of the tissue toevaluate efficacy of the treatment and adjust the same as needed.

It is yet another object of the present invention to provide a method oftissue ablation using electrical pulses that causes immediate cellularmembrane breakdown non-thermally so that sensitive tissue structures arespared and the intra-cellular and membrane proteins are spilled into theextracellular space without denaturing to be exposed to the body'simmune system in order to illicit a specific tumor immune response.

It is yet another object of the present invention to provide a method oftissue ablation that exposes non-denatured intra-cellular and membraneproteins to the immune system to illicit a specific tumor immuneresponse which can be modulated and enhanced by a variety of additionalimmune modulators.

According to the present invention, the above described and otherobjects are accomplished, by applying to undesirable tissue in the bodyan external electric field specifically configured to directly andcompletely disintegrate the cell membrane. Referred to as ElectricalMembrane Breakdown (EMB), application of an external oscillatingelectric field causes vibration and flexing of the cell membrane whichresults in a dramatic and immediate mechanical tearing or rupturing thecell membrane. EMB applies significantly higher energy levels than priorart methods to rupture the cell membrane rather than to electroporatethe cell membrane. Unlike prior art methods, EMB expels the entirecontents of the cell into the extracellular fluid and exposes internalcomponents of the cell membrane which induces an immunologic response bythe subject.

A system for generation of the electric field necessary to induce EMBincludes a bipolar pulse generator operatively coupled to a controllerfor controlling generation and delivery of the electrical pulsesnecessary to generate an appropriate electric field. The field isgenerated by therapeutic probes placed in proximity to the soft tissueor cancerous cells within the body of the subject and the bipolar pulsesare shaped, designed and applied to achieve that result in an optimalfashion. A temperature probe may be provided for temperature feedback tothe controller which is configured to control the signal outputcharacteristics of the signal generator. The EMB protocol calls for aseries of short and intense bi-polar electric to generate an oscillatingelectric field between the electrodes that induce a similarly rapid andoscillating buildup of transmembrane potential across the cell membrane.The built up charge applies a an oscillating and flexing force to thecellular membrane which upon reaching a critical value causes extensiverupture of the membrane and spillage of the cellular content. Inaddition to being bi-polar, the electric pulses preferably trace asquare wave form and are characterized by instant charge reversal thathave substantially no relaxation time between the positive and negativepolarities of the bi-polar pulse. Instant charge reversal pulses aresignificantly more effective in destruction of dielectric cell membranes

Important characteristic of the applied electric field include the fieldstrength (Volts/cm), frequency, polarity, shape, duration, number andspacing. Field strength (Volts/cm) is a function of both the appliedvoltage and the electrode spacing and is preferably in the range of1,500 V/cm to 10,000 V/cm absent thermal considerations. RF-EMB ablationis preferably performed by application of a series of not less than 100electric pulses in a pulse train so as to impart the energy necessary onthe target tissue without developing thermal issues in any clinicallysignificant way. The pulse duration is preferably from 100 to 1000 μs.The relationship between the duration and frequency of each pulsedetermines the number of instantaneous charge reversals experienced bythe cell membrane during each pulse. The duration of each inter pulseburst interval is determined by the controller 14 based on thermalconsiderations. Real time temperature feedback of the treatment site maybe provided to the controller by which the controller can modulatetreatment parameters to eliminate thermal effects as desired. Currentflow at the treatment site may also be monitored for this purpose.

The EMB ablation method is carried out by first identifying the locationof the soft tissue within the subject to be ablated by medical imagingtechniques such as CT or MRI or other means. A preferred position andspacing of the electrodes relative to the target tissue is determinedand from 1 to 6 needle electrodes connected to the controller and signalgenerator are inserted into position in and around the treatment site.Placement and positioning of the electrodes is confirmed by medicalimaging and the pulse generator is activated to apply electrical pulsesto the electrodes to generate the treatment field thereby causingelectrical membrane breakdown of cells in the soft tissue.

Electrical membrane breakdown causes immediate spillage of allintracellular components of the ruptured cells into an extracellularspace and exposes the internal constituent parts of the cell and cellmembrane including antigens which induce an immunologic response todestroy and remove this and like material in the body of the subject.The immunologic response can be enhanced by administration of agentsthat increase the immunologic response process including drugs.Electrical membrane breakdown causes immediate, visually observabletissue change, cellular membrane destruction and cell death such thatthe method may include the biopsy of a portion of the treated targettissue to verify treatment efficacy immediately after completion of thetreatment while the patient is still in position for additionaltreatment. In other embodiments needle probes placed in criticaltreatment locations could monitor various parameters by means ofchemical or spectroscopic means related to the immediate destruction andspillage of the intra-cellular contents also to verify treatmentefficacy. In some situation, the mode of treatment may be switched fromEMB to thermal ablation without removal or repositioning of theelectrodes by reconfiguring the signal generated by the pulse generatorto increase the tissue temperature at the electrodes according to knownRF thermal techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a cell membrane pore.

FIG. 2 is a diagram of cell membrane pore formation by a prior artmethod.

FIG. 3 is a comparison of a prior art charge reversal with an instantcharge reversal according to the present invention.

FIG. 4 is a square wave from instant charge reversal pulse according tothe present invention.

FIG. 5 is a diagram of the forces imposed on a cell membrane as afunction of electric field pulse width according to the presentinvention.

FIG. 6 is a diagram of a prior art failure to deliver prescribed pulsesdue to excess current.

FIG. 7 is a schematic diagram of a feedback loop according to thepresent invention by which a controller reduces an applied signalvoltage to keep the current amperage at or below a maximum.

FIG. 8 is a diagram of a reduction in applied signal voltage uponreaching a maximum current level to permit continued signal deliveryaccording to the present invention.

FIG. 9 is a schematic diagram of a pulse generation and delivery systemfor application of the method of the present invention.

FIG. 10 is a diagram of the parameters of a partial pulse trainaccording to the present invention.

FIG. 11 is a chart of exemplary treatment protocol parameters accordingto the present invention.

FIG. 12 is a diagram of the parameters of exemplary treatment protocolnumber 1.

FIG. 13 is a diagram of the parameters of exemplary treatment protocolnumber 2.

FIG. 14 is a diagram of the parameters of exemplary treatment protocolnumber 3.

FIG. 15 is a diagram of the parameters of exemplary treatment protocolnumber 4.

DETAILED DESCRIPTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not limit the scope of the invention.

Irreversible electroporation as a tissue ablation method is welldeveloped with commercially manufactured equipment such as the NanoKnifeby AngioDynamics (Latham, N.Y.) available on the market. As described,this ablation technique utilizes high electric field strengths, withinspecific parameters, to induce irreversible electroporation of the cellmembrane resulting in eventual cell death due to loss of homeostasis andapoptosis. The present invention also describes methods for ablatingcells within the body of a subject utilizing high frequency and highstrength electric fields but does so through the entirely differentprocess of Electrical Membrane Breakdown (EMB) using very differentenergy characteristics. Electrical Membrane Breakdown is the applicationof an external oscillating electric field to cause vibration and flexingof the cell membrane which results in a dramatic and immediatemechanical tearing, disintegration or rupturing of the cell membrane.Unlike IRE, in which nano-pores are created in the cell membrane butthrough which little or no content of the cell is released, EMBcompletely tears open the cell membrane such that the entire contents ofthe cell are expelled into the extracellular fluid, and internalcomponents of the cell membrane itself are exposed.

The present invention relies on the interaction of an applied electricfield with the transmembrane potential but its similarity to IRE endsthere. EMB applies significantly higher energy levels by specificallyconfigured electric field profiles to directly and completelydisintegrate the cell membrane rather than to electroporate the cellmembrane. Others have demonstrated that the energy levels required forEMB is 100 times greater than for IRE using the same pulseconfigurations (pulse number and voltage density) delivered by currentlyavailable IRE equipment and protocols. The inability of current IREmethods and energy protocols to deliver the energy necessary to causeEMB explains why pathologic examination of IRE treated specimens hasnever shown the pathologic characteristics of EMB and is a criticalreason why EMB had not until now been recognized as an alternativemethod of cell destruction.

FIG. 9 is a schematic diagram of a system 10 for generation of theelectric field necessary to induce EMB of cells 11 within a patient 12.The system 10 includes a bipolar pulse generator 16 operatively coupledto a controller 14 for controlling generation and delivery to thetherapeutic probe or probes 20 (two are shown) of the electrical pulsesnecessary to generate an appropriate electric field to achieve EMB. Thetherapeutic probes are placed in proximity to the soft tissue orcancerous cells 11 which are intended to be ablated through the processof EMB and the bipolar pulses are shaped, designed and applied toachieve that result in an optimal fashion. A temperature probe 22 may beprovided for percutaneous temperature measurement and feedback to thecontroller of the temperature at or near the electrodes. The controllermay preferably include an onboard digital processor and a memory and maybe a general purpose computer system, programmable logic controller orsimilar digital logic control device. The controller is preferablyconfigured to control the signal output characteristics of the signalgeneration including the voltage, frequency, shape, polarity andduration of pulses as well as the total number of pulses delivered in apulse train and the duration of the inter pulse burst interval.

With reference to FIG. 9, the EMB protocol calls for a series of shortand intense bi-polar electric pulses delivered from the pulse generatorthrough one or more therapeutic probes 20 (electrodes) inserted directlyinto, or placed around the target tissue 11. The bi-polar pulsesgenerate an oscillating electric field between the electrodes thatinduce a similarly rapid and oscillating buildup of transmembranepotential across the cell membrane. The built up charge applies anoscillating and flexing, force to the cellular membrane which uponreaching a critical value causes rupture of the membrane and spillage ofthe cellular content. Bipolar pulses are more lethal than monopolarpulses because the pulsed electric field causes movement of chargedmolecules in the cell membrane and reversal in the orientation orpolarity of the electric field causes a corresponding change in thedirection of movement of the charged molecules and of the forces actingon the cell. The added stresses that are placed on the cell membrane byalternating changes in the movement of charged molecules createadditional internal and external changes that cause indentations,crevasses, rifts and irregular sudden tears in the cell membrane causingmore extensive, diverse and random damage and disintegration of the cellmembrane.

With reference to FIG. 4, in addition to being bi-polar, the preferredembodiment of electric pulses is one for which the voltage over timetraces a square wave form and is characterized by instant chargereversal pulses (ICR). A square voltage wave form is one that maintainsa substantially constant voltage of not less than 80% of peak voltagefor the duration of the single polarity portion of the trace, exceptduring the polarity transition. An instant charge reversal pulse is apulse that is specifically designed to ensure that substantially norelaxation time is permitted between the positive and negativepolarities of the bi-polar pulse. That is, the polarity transitionhappens virtually instantaneously.

The destruction of dielectric cell membranes through the process ofElectrical Membrane Breakdown is significantly more effective if theapplied voltage pulse can transition from a positive to a negativepolarity without delay in between. Instant charge reversal preventsrearrangement of induced surface charges resulting in a short state oftension and transient mechanical forces in the cells, the effects ofwhich are amplified by large and abrupt force reversals. Alternatingstress on the target cell that causes structural fatigue is thought toreduce the critical electric field strength required for EMB. The addedstructural fatigue inside and along the cell membrane results in orcontributes to physical changes in the structure of the cell. Thesephysical changes and defects appear in response to the force appliedwith the oscillating EMB protocol and approach dielectric membranebreakdown as the membrane position shifts in response to theoscillation, up to the point of total membrane rupture and catastrophicdischarge. This can be analogized to fatigue or weakening of a materialcaused by progressive and localized structural damage that occurs when amaterial is subjected to cyclic loading, such as for example a metalpaper clip that is subjected to repeated bending. The nominal maximumstress values that cause such damage may be much less than the strengthof the material under ordinary conditions. The effectiveness of thiswaveform compared to other pulse waveforms can save up to ⅕ or ⅙ of thetotal energy requirement.

With reference to FIG. 10, another important characteristic of theapplied electric field is the field strength (Volts/cm) which is afunction of both the voltage 30 applied to the electrodes by the pulsegenerator 16 and the electrode spacing. Typical electrode spacing for abi-polar, needle type probe might be 1 cm, while spacing betweenmultiple needle probe electrodes can be selected by the surgeon andmight typically be from 0.75 cm to 1.5 cm. A pulse generator forapplication of the present invention is capable of delivering up to a 10kV potential. The actual applied field strength will vary over thecourse of a treatment to control circuit amperage which is thecontrolling factor in heat generation, and patient safety (preventinglarge unanticipated current flows as the tissue impedance falls during atreatment). Where voltage and thus field strength is limited by heatingconcerns the duration of the treatment cycle may be extended tocompensate for the diminished charge accumulation. Absent thermalconsiderations, a preferred field strength for EMB is in the range of1,500 V/cm to 10,000 V/cm.

With continued reference to FIG. 10, the frequency 31 of the electricsignal supplied to the electrodes 20, and thus of the field polarityoscillations of the resulting electric field, influences the totalenergy imparted on the subject tissue and thus the efficacy of thetreatment but are less critical than other characteristics. A preferredsignal frequency is from 14.2 kHz to less than 500 kHz. The lowerfrequency bound imparts the maximum energy per cycle below which nofurther incremental energy deposition is achieved. With reference toFIG. 5, the upper frequency limit is set based on the observation thatabove 500 kHz, the polarity oscillations are too short to develop enoughmotive force on the cell membrane to induce the desired cell membranedistortion and movement. More specifically, at 500 kHz the duration of asingle full cycle is 2 μs of which half is of positive polarity and halfnegative. When the duration of a single polarity approaches 1 μs thereis insufficient time for charge to accumulate and motive force todevelop on the membrane. Consequently, membrane movement is reduced oreliminated and EMB does not occur. In a more preferred embodiment thesignal frequency is from 100 kHz to 450 kHz. Here the lower bound isdetermined by a desire to avoid the need for anesthesia orneuromuscular-blocking drugs to limit or avoid the muscle contractionstimulating effects of electrical signals applied to the body. The upperbound in this more preferred embodiment is suggested by the frequency ofradiofrequency thermal ablation equipment already approved by the FDA,which has been deemed safe for therapeutic use in medical patients.

In addition to controlling the pulse amplitude 30, frequency 31,polarity and shape provided by the pulse generator 16, the logiccontroller 14 controls the number of pulses 32 to be applied in thetreatment series or pulse train, the duration of each pulse 32, and theinter pulse burst delay 33. Although only two are depicted in FIG. 10due to space constraints, RF-EMB ablation is preferably performed byapplication of a series of not less than 100 electric pulses 32 in apulse train so as to impart the energy necessary on the target tissue 11without developing thermal issues in any clinically significant way. Thewidth of each individual pulse 32 is preferably from 100 to 1000 μs withan inter pulse burst interval 33 during which no voltage is applied inorder to facilitate heat dissipation and avoid thermal effects. Therelationship between the duration of each pulse 32 and the frequency 31(period) determines the number of instantaneous charge reversalsexperienced by the cell membrane during each pulse 32. The duration ofeach inter pulse burst interval 33 is determined by the controller 14based on thermal considerations. In an alternate embodiment the system10 is further provided with a temperature probe 22 inserted proximal tothe target tissue 11 to provide a localized temperature reading at thetreatment site to the controller 14. The temperature probe 22 may be aseparate, needle type probe having a thermocouple tip, or may beintegrally formed with or deployed from one or more of the needleelectrodes. With temperature feedback in real time, the controller canmodulate treatment parameters to eliminate thermal effects as desired bycomparing the observed temperature with various temperature set pointsstored in memory. More specifically, the controller can shorten orincrease the duration of each pulse 32 to maintain a set temperature atthe treatment site to, for example, create a heating (high temp) for theneedle tract to prevent bleeding or to limit heating (low temp) toprevent any coagulative necrosis. The duration of the inter pulse burstinterval can be modulated in the same manner in order to eliminate theneed to stop treatment and maximizing the deposition of energy toaccomplish RF-EMB. Pulse amplitude 30 and total number of pulses in thepulse train may also be modulated for the same purpose and result.

In yet another embodiment, the controller may monitor or determinecurrent flow through the tissue during treatment fir the purpose ofavoiding overheating while yet permitting treatment to continue byreducing the applied voltage. Reduction in tissue impedance duringtreatment due to charge buildup and membrane rupture can cause increasedcurrent flow which engenders additional heating at the treatment site.With reference to FIG. 6, prior treatment methods have suffered from aneed to cease treatment when the current exceeds a maximum allowablesuch that treatment goals are not met. As with direct temperaturemonitoring, the present invention can avoid the need to stop treatmentby reducing the applied voltage and thus current through the tissue tocontrol and prevent undesirable clinically significant thermal effects.Modulation of pulse duration and pulse burst interval duration may alsobe employed by the controller 11 for this purpose as described.

With reference to FIG. 1I, four exemplary RF-EMB treatment protocols aredetailed. With additional reference to FIG. 12, in protocol 1, a pulsetrain of 83 pulses 32 each a 10 ms duration is applied at 600 volts toelectrodes spaced at 1 cm resulting in a field strength of 600 V/cmbetween the electrodes. In this example the applied pulses are bipolarwith a frequency of 125 kHz with a pulse width of 10 ms, such that thetotal energy applied over the 0.83 seconds duration of the pulse trainwas 10.38 mJ. These treatment models and the total energy delivered werereferenced from work describing energy parameters used for membranebreakdown of algae by, Foltz, G., Algae Lysis With Pulsed ElectricFields, California State Polytechnic University, San Luis Obispo 2012,downloaded from http://digitalcommons.-calpoly.edu/theses/732/. Foltzdemonstrated this energy requirement using unipolar pulses, without theadvantage of instant charge reversal pulses, making this the worst casescenario for energy requirements to produce EMB.

With reference to FIG. 13, in protocol 2 EMB is achieved by a pulsewidth decreased to 200 μs and pulse train extended to 2490 pulses in a10 kV/cm field for a total treatment time of 0.49 seconds. The totalapplied energy is again 10.38 mJ. With reference to FIG. 14, in protocol3 additional pulses above the initially targeted 2490 are added by thecontroller 11 to compensate for reduction in voltage/field strengthduring treatment based on feedback from the treatment site. Withreference to FIG. 15, in protocol 4 the additional pulses above theinitially targeted 2490 are added to compensate for loss of efficiencyresulting from the 250 kHz signal as compared to the 125 kHz signalfrequency in the previous exemplary protocols.

The method of ablating undesirable soft tissue of the present inventionis carried out by first identifying the location of the soft tissuewithin the subject to be ablated. Tissue identification may be done byknown medical imaging techniques such as ultrasound, CT or MRI. Thetarget soft tissue may or may not be a malignancy but rather need onlybe tissue that is undesirable in its present location for some reason.After identification of the target tissue, the preferred position andspacing of the electrodes relative to target soft tissue is determinedbased on the location and shape of the tissue to be ablated, the shapeand location of adjacent structures, the dielectric constant and theconductivity of the target and surrounding soft tissue. Typically from 1to 6 needle type probe electrodes are used. The electrodes areintroduced into position in and around the treatment and connected to acontroller for controlled delivery of the electric pulses for fieldgeneration and treatment. The probe electrodes may include a temperaturesensor such as a thermocouple for reading and signaling to thecontroller the local temperature at or near the electrode. Placement andpositioning of the electrodes may preferably be confirmed by medicalimaging. The pulse generator is activated by the controller to applyelectrical pulses to the electrodes to generate the treatment field asdescribed above thereby causing electrical membrane breakdown of some orall of cells of said soft tissue.

Electrical membrane breakdown, unlike IRE or thermal ablationtechniques, causes immediate spillage of all intracellular components ofthe ruptured cells into an extracellular space and exposes the internalconstituent part of the cell membrane to the extracellular space. Theintracellular components include cellular antigens and the internalconstituent parts of the cell membrane include antigens specific to thecell membrane which induce an immunologic response to destroy and removethis and like material in the body of the subject. Like material may beother material in the body of the subject having the same cellularantigens or cell membrane specific antigens at locations remote from thetreatment site including metastatic tissue. However, the human body alsohas natural defense systems for tumors which prevent destruction and/orremoval of the tumor in some cases. One of these operates via aninhibitory signal, which presents itself to the body's cytotoxic Tlymphocytes (CTLs), the cells in the body that recognize and destroycancer cells, and binds to the cytototoxic T lymphocyte-associatedantigen 4 (CTLA-4) receptor, turning off the cytotoxic reaction that mayotherwise destroy the cancer cell.

Thus, according to another embodiment of the present invention, theimmunologic response of RF-EMB is enhanced by administration of drugsthat increase the immunologic response process including drugs whichblock inhibition of the CTLA-4 inhibitory signal of cytotoxiclymphocytes, or that bind to the S100-A9 protein, which is involved inmodulating regulatory myeloid cell functions. An example of the formerdrug type is Ipilimumab (marketed as Yervoy®). An example of the latteris Tasquinimod. Such drugs can be administered by any means, includingwithout limitation, intravenously, orally or intramuscularly and mayfurther be injected directly into or adjacent to the target soft tissueimmediately before or after applying the EMB electric field or a setnumber of days before or after an RF-EMB treatment, as described in thesample treatment protocols below. Such immunologic response enhancingdrug may be comprised also of autologous dendritic cells. For example,Sipuleucel-T (marketed as Provenge®) therapy uses autologous patientdendritic cells activated with prostatic acid phosphatase (PAP) andinfused back into the patient's system. Another relevant immunologicdrug is pembrolizumab, which works by blocking a protein known asProgrammed Death receptor (PD-1), or a related protein known as PD-L1,both of which are used by tumors as a defense to tumor-fighting cells.Yet another relevant immunologic drug is cyclophosphamide, whichdepresses regulatory T cells and interfere with DNA replication. Manyimmunologic drugs such as those described herein are effective againstone or a small handful of cancer types, but are not effective, inisolation, against all cancer types for which this class of drugs wasdesigned to be used.

Combining RF-EMB treatment with the administration of an immunologicdrug such as those described above leaves the target cells' antigensintact and exposed to the external environment, allowing them to reactwith the patient's immune system, all of which aids the functioning ofthe immunologic drug. The combination treatment may aid in the treatmentof patients with one of two distinct disease pathologies. In a firstembodiment, comprising a method for treating a patient with a primarycancerous tumor and a high likelihood of micrometastatic disease, RF-EMBmay be applied to cause direct destruction of the primary tumor precededor followed by the administration of a immunologic drug regimen designedto interact cooperatively with the intact antigens which have beenexposed as a result of the RF-EMB treatment. The immunologic drug chosenmay be one that blocks the inhibitory response that may otherwiseprevent the patient's body from recognizing and destroying the RF-EMBtarget cells and others having the same cellular antigens (i.e.,micrometastatic growths) as a result of the RF-EMB treatment. In asecond embodiment, comprising a method for treating a patient havingadvanced metastatic disease, RF-EMB treatment may be administered atmidpoints of an ongoing treatment plan utilizing an immunologic drug asdescribed above. Under this embodiment. RF-EMB treatments enhance theeffectiveness of the immunologic drug by exposing unique cellularantigens to the patient's immune system.

Three sample treatment protocols for the use of RF-EMB in conjunctionwith the administration of an immunologic drug are now described. InExample 1, 300 mg/m² of cyclophosphamide are administered intravenouslyto the patient on Day 1 of treatment. On Day 3, the patient receivesRF-EMB treatment according to one of the four protocols described abovewith reference to FIG. 11. Beginning two weeks after the RF-EMBtreatment and lasting until week 26 following RF-EMB treatment, 25 mg ofcyclophosphamide is administered to the patient orally for six cycles,each cycle comprising four weeks, wherein the patient receives an oraldose of cyclophosphamide twice daily in cycles of seven days on (whereinthe drug is administered), seven days off (wherein no drug isadministered). In Example 2, the patient is treated on Day 1 with RF-EMBtreatment according to one of the four protocols described above withreference to FIG. 11. Also on Day 1, the patient is given 3 mg/kg ofipilimumab intravenously over the course of 90 minutes. The patient thenreceives an additional three doses of ipilimumab, 3 mg/kg intravenously,each dose separated by a period of three weeks. In Example 3, 300 mg/m²of cyclophosphamide are administered intravenously to the patient on Day1 of treatment. On Day 3 of treatment, the patient receives RF-EMBtreatment according to one of the four protocols described above withreference to FIG. 11, with the addition of the injection of autologousdendritic cells directly into the target tumor. Beginning two weeksafter the RF-EMB treatment and lasting until week 26 following RF-EMBtreatment, 25 mg of cyclophosphamide is administered to the patientorally for six cycles, each cycle comprising four weeks, wherein thepatient receives an oral dose of cyclophosphamide twice daily in cyclesof seven days on (wherein the drug is administered), seven days off(wherein no drug is administered).

Electrical membrane breakdown causes immediate, visually observabletissue change, cellular membrane destruction and cell death. As aresult, the method may include the biopsy of a portion of the treatedtarget tissue to verify treatment efficacy immediately after completionof the treatment while the patient is still in position for additionaltreatment. Additional treatment may be immediately administered based onthe biopsy result and visual determination of treatment efficacy.

Alternatively, because the intracellular environment comprises a uniquechemical composition, such as high potassium and uric acidconcentrations, spillage of the cell contents can now be detected bymethods such as placing one or more needle probes into criticallocations of the treatment area to measure chemical levels usingchemical reagents, electrical impedance or resistance measurements, pHmeasurements, spectroscopy, or the like. Moreover, a device such as amicroneedle sensor, comprising one or more sensors capable of measuringthe above qualities integrated into or inserted through the hollow coreof a microneedle, may be inserted at one or more predetermined locationsin the treatment area during an RF-EMB procedure to measure cellularspillage via extracellular chemical composition in real time.

According to this method, in a preferred embodiment, a hollow needlehaving at least one dimension of less than 1 millimeter (known as amicroneedle) is outfitted with one or more sensors by inserting thesensor(s) through the hollow center of the needle. The sensor(s) may beone or more of the types described above, including but not limited to apH sensor, a lactate sensor, a glucose sensor, an electrical impedancesensor, a potassium sensor, and/or a uric acid sensor. Multiple suchsensors may be bundled together or a single sensor could be used whichmeasures one or more of the relevant properties. In an alternativeembodiment, the sensor may be a spectrometer. Most preferably, one ormore sensor-containing microneedles are inserted into the selectedtreatment area immediately prior to the application of RF-EMB treatment,and remain inserted into the treated tissue for the entire duration ofthe treatment session. Readings from the sensors may be measured by anymeans known in the art. Such a method has the added benefit of allowingthe treatment provider to observe and quantify the level of target celldestruction, and thereby treatment efficacy, in real time and in vivo.By contrast, prior art, thermal ablation methods or non thermal ablationmethods such as IRE lack this capability in that they do not cause ameasurable amount of the cellular contents to be spilled into theextracellular area immediately, resulting instead in thermal necrosis ortargeted apoptotic cell death which destroys the cell and its contentsbefore any of the cellular contents are exposed for measurement. Thus,prior art ablation methods often required a biopsy of the treated areato determine treatment efficacy, which cannot be completed until thetermination of the treatment.

According to this preferred embodiment, treatment parameters and/orlocation(s) may be monitored and/or adjusted in real time based on thereal time measured levels of cellular spillage during the treatmentprocess. In addition, or alternatively, measurements of the cellularcontents as described herein may be taken before, after, or betweenphases of treatment without the need to subject the patient to a biopsyor other invasive procedure to measure treatment efficacy. Measurementtechniques for cellular contents are not limited to those describedherein, but may be carried out by any means known in the art ofmeasuring chemical compositions of a targeted treatment area in vivoand/or in real time.

In yet another alternate embodiment of the present invention, with orwithout intermediate biopsy and visual observation for efficacy, themode of treatment according to the present invention may be switchedfrom EMB to thermal ablation without removal or repositioning of theelectrodes. A switch to thermal ablation may be desirable to controlbleeding at the tissue site or for direct destruction of undesirabletissue in concert with the RF-EMB. The switch may occur within a singlepulse train by operation of the controller, or may be accomplished by asecond or additional pulse train directed to RF thermal ablation only.The switch is accomplished by reconfiguring the signal generated by thepulse generator to increase the tissue temperature at the electrodesaccording to known RF thermal techniques.

Having now fully set forth the preferred embodiment and certainmodifications of the concept underlying the present invention, variousother embodiments as well as certain variations and modifications of theembodiments herein shown and described will obviously occur to thoseskilled in the art upon becoming familiar with said underlying concept.It is to be understood, therefore, that the invention may be practicedotherwise than as specifically set forth in the appended claims.

INDUSTRIAL APPLICABILITY

Studies estimate that cancer kills approximately 20,000 people worldwideper day. Many casualties could be avoided and the quality of life couldbe improved for many patients with more effective, minimally invasivemethods treatment of cancerous tumors and other conditions resulting inunwanted soft tissue. Minimally invasive treatments capable of assistinga patient's own immune system in attacking and removing unwanted orcancerous tissue within the patient's body would further aid in savinglives and improving patient quality of life. What is needed is aminimally invasive method of removal of unwanted soft tissue, such ascancerous tumors. The present invention is an innovative method ofablating unwanted soft tissue within a patient's body that hasapplicability to many types of cancerous as well as non-canceroustissue, that significantly improves effectiveness of performing such aprocedure, and that further provides a means to directly measure theefficacy of such procedures in vivo and simultaneous with treatment.

We claim:
 1. A method of ablating undesirable soft tissue in a livingsubject, comprising: introducing at least one electrode to a positionwithin said subject, wherein said at least one electrode is electricallyconnected to a controller for controlling the delivery of electricpulses to said electrode, said controller comprising an electric pulsegenerator; applying to said soft tissue an electric field of between1,500 V/cm and 10,000 V/cm and having a frequency of between 14.2 kHzand 500 kHz, including delivering, from the electric pulse generator tothe at least one electrode, at least one bi-polar electric pulse; and byapplication of the electric field, causing electrical membrane breakdownof a cell membrane of a plurality of cells of said soft tissue to causedestruction of the cell membrane, immediate spillage of intracellularcomponents into an extracellular space, and exposure of an internalconstituent part of said cell membrane to said extracellular space. 2.The method of claim 1 wherein a voltage of said bi-polar electric pulsesis from 0.5 kV to 10 kV.
 3. The method of claim 2 wherein said voltageof said bi-polar electric pulse is characterized by an instant chargereversal, between the positive and negative charge of each cycle.
 4. Themethod of claim 1 wherein a frequency of said electric field is from 100kHz to 450 kHz.
 5. The method of claim 1 wherein said voltage over timeof each set of bi-polar electric pulses traces a square waveform for apositive and negative component of a polarity oscillation.
 6. The methodof claim 1 wherein the duration of each set of bi-polar electric pulsesis from 100-1000 μs.
 7. The method of claim 1, comprising determiningthe position of said at least one electrode relative to said softtissue, including estimating or measuring the dielectric constant andthe conductivity of said soft tissue.
 8. The method of claim 1 whereineach set of bi-polar electric pulses includes at least 100 bi-polarelectric pulses.
 9. The method of claim 1, further comprisingconfiguring said series of bi-polar electric pulses delivered to said atleast one electrode from said pulse generator such that said electricfield causes no clinically significant thermal damage to said softtissue.
 10. The method of claim 9, comprising configuring said series ofelectric pulses such that said electric field causes a temperature ofsaid soft tissue to rise to not more than 50 degrees Celsius.
 11. Themethod of claim 1, further comprising applying to said soft tissue asecond electric field, including delivering from said pulse generator tosaid at least one electrode a second series of electric pulses.
 12. Themethod of claim 11, further comprising configuring said second series ofelectric pulses such that the applied second electric field causesclinically significant thermal damage to said soft tissue.
 13. Themethod of claim 11 wherein said second series of bi-polar electricpulses comprises multiple sets of bi-polar electric pulses, each set ofbi-polar electric pulses including not less than 100 bi-polar electricpulses configured so that said second electric field is sufficient tocause electrical membrane breakdown of said cell membrane of a pluralityof cells of said soft tissue.
 14. The method of claim 11 wherein saidsecond series of bi-polar electric pulses is configured so that saidsecond electric field causes a temperature of at least a portion of saidtissue to exceed 50 degrees Celsius.
 15. The method of claim 14 whereinsaid second electric field causes a temperature of at least a portion ofsaid tissue to exceed 60 degrees Celsius but not to exceed 95 degreesCelsius.
 16. The method of claim 1, further comprising delivering fromsaid pulse generator to said at least one electrode a second series ofelectric pulses configured to cause a temperature of at least a portionof said soft tissue to exceed 50 degrees Celsius.
 17. The method ofclaim 1, wherein one or more of a tissue change, cellular membranedestruction and cell death are visually observable in a sample of saidundesirable soft tissue taken immediately after applying said electricfield to said soft tissue; and the method further comprising: afterapplying said electric field to said soft tissue by delivering saidseries of bi-polar electric pulses, immediately biopsying a portion ofsaid soft tissue to determine an efficacy of said ablation; if saidefficacy exceeds a pre-determined threshold, ceasing said ablation ofundesirable soft tissue; and if said efficacy does not exceed saidpre-determined threshold, delivering from said pulse generator to saidat least one electrode a second series of bi-polar electric pulses so asto apply to said soft tissue a second electric field.
 18. The method ofclaim 1, further comprising introducing a temperature probe proximal tosaid at least one electrode, said probe operatively connected to saidcontroller and configured to report a temperature reading to saidcontroller, said controller configured to control the varying of atleast one characteristic of said series of bi-polar electric pulses inresponse to said reported temperature.
 19. The method of claim 18,wherein said temperature probe is a thermocouple.
 20. The method ofclaim 18, wherein said temperature probe is integral to said at leastone electrode and introduced therewith.
 21. The method of claim 18,wherein each set of bi-polar electric pulses includes not less than 100bi-polar pulses; and the method further comprising: storing in a memoryof said controller at least one temperature set-point; and altering, bysaid controller, for at least a portion of said series of bi-polarelectric pulses, at least one of a pulse duration, a duration of saidinterval, and a total number of bi-polar electric pulses in one or moreof the sets of bi-polar pulses in said series in response to saidtemperature reading reported to said controller from said probeexceeding said set-point.
 22. The method of claim 21, further comprisingaltering at least one of said pulse duration, the duration of saidinterval, and the total number of bi-polar electric pulses in responseto said temperature reading reported by said probe falling below saidset-point.
 23. The method of claim 22, wherein altering at least one ofsaid pulse duration, the duration of said interval, and the total numberof pulses comprises increasing the duration of said interval.
 24. Themethod of claim 21, wherein altering at least one of said pulseduration, the duration of said interval, and the total number of pulsescomprises reducing said pulse duration.
 25. The method of claim 24,wherein altering at least one of said pulse duration, the duration ofsaid interval, and the total number of pulses comprises reducing thenumber of pulses in one or more of the sets of bi-polar pulses in saidseries.
 26. The method of claim 1, further comprising storing in amemory of the controller at least one maximum current set-point;determining by said controller a current through said at least oneelectrode; and reducing said voltage of said at least one of saidbi-polar electric pulses delivered to said at least one electrode whensaid current equals said maximum current set-point.
 27. The method ofclaim 1 wherein electrical membrane breakdown of the cell membrane ofthe plurality of cells causes spillage of intracellular components intoan extracelluar space, and wherein an immunologic process of saidsubject is activated to remove said intracellular components and aninternal constituent part of said cell membrane from said extracellularspace.
 28. The method of claim 27 wherein said intracellular componentscomprise a cellular antigen and said internal constituent part of saidcell membrane comprises a cell membrane specific antigen; and whereinsaid immunologic process comprises removal of undesirable soft tissue ata second location in said living subject, said undesirable soft tissueat said second location having one or more of said cellular antigen andsaid cell membrane specific antigen, said undesirable soft tissue atsaid second location in said living subject not having undergone saidmethod of ablating undesirable soft tissue.
 29. The method of claim 27wherein said intracellular components comprise a cellular antigen andsaid internal constituent part of said cell membrane comprises anantigen specific to said cell membrane; and the method furthercomprising administering to said subject an immunologic responseenhancing drug to increase said immunologic process.
 30. The method ofclaim 29 wherein said immunologic response enhancing drug is configuredto block inhibition of the CTLA-4 inhibitory signal of cytotoxiclymphocytes.
 31. The method of claim 29 comprising administering saidimmunologic response enhancing drug by one of intravenously, orally andintramuscularly.
 32. The method of claim 29 comprising injecting saidimmunologic response enhancing drug directly into or adjacent toundesirable soft tissue before or after applying the electric field tosaid soft tissue.
 33. The method of claim 29 wherein said immunologicresponse enhancing drug comprises autologous dendritic cells.
 34. Themethod of claim 29 wherein said immunologic response enhancing drug isconfigured to bind to S100A9 and to modulate regulatory mycloid cellfunctions.
 35. The method of claim 29 wherein said immunologic responseenhancing drug is configured to block a protein selected from the groupcomprising PD-1 and PD-L1.
 36. The method of claim 1, furthercomprising: inserting one or more sensors into said soft tissue withinsaid subject; obtaining a plurality of measurements from said one ormore sensors simultaneously with applying to said soft tissue theelectric field sufficient to cause electrical membrane breakdown of thecell membrane of the plurality of cells; and determining, based on saidmeasurements, a treatment efficacy of said method of ablatingundesirable soft tissue in a living subject.
 37. The method of claim 36,wherein at least one of said one or more sensors is chosen from thegroup comprising a pH sensor, a lactate sensor, a glucose sensor, anelectrical impedance sensor, a potassium sensor, a uric acid sensor, anda spectrometer.
 38. The method of claim 36, further comprising alteringone or more parameters of said electric field based on said treatmentefficacy.
 39. The method of claim 1, wherein said series of bi-polarelectric pulses comprises: multiple sets of bi-polar pulses, and aninterval separating each set of bi-polar pulses from the successive setof bi-polar pulses, in which during each interval, substantially novoltage is applied to said at least one electrode.
 40. A method ofablating undesirable soft tissue in a living subject, comprising:determining based on a tissue type of said soft tissue a minimum energyprofile necessary to be applied to a cell of said soft tissue to causecell membrane rupture by electrical membrane breakdown; determining aposition of at least one electrode relative to said soft tissue;introducing said at least one electrode to said position within saidsubject, said electrode electrically connected to a controller forcontrolling the delivery of electric pulses to said electrode, saidcontroller comprising an electric pulse generator; determining based onsaid minimum energy profile and said position of said at least oneelectrode an electric field strength to be applied to said soft tissue;determining based on said electric field strength a bi-polar electricpulse train profile having not less than 100 pulses, said pulse trainprofile characterized by a pulse number, pulse duration and inter pulseburst intervals, said pulses each having a frequency and a voltage, saidvoltage characterized by an instantaneous reversal of polarity;delivering from said pulse generator to said at least one electrode bysaid controller a series of electric pulses according to said electricpulse train profile whereby a pulsed electric field is generated, saidfield applying sufficient energy to a plurality of said cells of saidsoft tissue to cause cell death by electrical membrane breakdown.
 41. Amethod of increasing an immunologic response in a living subject,comprising: performing a non-thermal ablation treatment on undesirablesoft tissue within said subject, said non-thermal ablation treatmentcomprising applying to said soft tissue an electric field sufficient tocause electrical membrane breakdown of a cell membrane of a plurality ofcells of said soft tissue to cause immediate spillage of allintracellular components into an extracellular space and exposure of aninternal constituent part of said cell membrane to said extracellularspace, wherein the electric field comprises a series of bi-polarelectric pulses that is characterized by an instant charge reversalbetween a positive and negative polarity of each cycle, and wherein theseries of bi-polar electric pulses comprises: multiple sets of bi-polarpulses; and an interval separating each set of bi-polar pulses from thesuccessive set of bi-polar pulses, in which during the interval,substantially no voltage is applied to the at least one electrode; andadministering an immunologic response enhancing drug to said subject.42. The method of claim 41, wherein performing a non-thermal ablationtreatment comprises introducing at least one electrode to a positionwithin said subject, said electrode electrically connected to acontroller for controlling the delivery of electric pulses to saidelectrode, said controller comprising an electric pulse generator; andapplying said electric field by delivering from said pulse generator tosaid at least one electrode a series of bi-polar electric pulses. 43.The method of claim 41, wherein said immunologic response enhancing drugis configured to block a protein selected from the group comprising PD-1and PD-L1.
 44. The method of claim 41, wherein said immunologic responseenhancing drug is configured to block inhibition of the CTLA-4inhibitory signal of cytotoxic lymphocytes.